Process Development and cGMP Manufacturing of a Recombinant Ricin Vaccine: an Effective and Stable Recombinant Ricin A-Chain Vaccine—RVE<i>c</i>TM

Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A-chain (RTA), which is an N-glycosidase enzyme, and the B-chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1-33/ 44-198 protein, herein denoted RVEa TM) expressed in Escherichia coli using a codon-optimized gene was shown to be non-toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale-up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)-compliant production of RVEc TM at the 40 L scale. The average yield of the final purified bulk RVEc TM is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc TM was [99% pure by three HPLC methods and SDS-PAGE. The intact mass and peptide mapping analysis of RVEc TM confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc TM was immuno-protective against lethal ricin challenge and elicited neutralizing anti-ricin antibodies in 95–100% of the vaccinated mice. Introduction The ricin toxin is a 64 kilodalton (kDa) protein produced by the seeds of the castor oil plant (Ricinus communis). The mechanism of action of this opportunistic ribotoxin has been thoroughly studied. 1,2 The holotoxin is a glycosylated hetero-dimeric protein that is composed of two chains, A and B, joined by a single disulfide bond. 3–5 The A-chain (RTA) is an enzyme that inactivates the 28S ribosomal ribonucleic acid (rRNA) by hydrolytic cleavage of the N-glycosidic bond of the essential adenine nucleotide (A4324) of the ribo-some. 6,7 The depurination site in the rRNA is part of a conserved sequence present in nearly all eukaryotic cells and it is known as the sarcin-ricin loop. 8 The B-chain (RTB) is a lectin that binds to galactose moieties presented by cell-surface glycopeptides or glycolipids, inducing endocytosis of the holotoxin. The combination of the internalization by the toxin and the cytosolic activity of the A-chain (kcat/Km $10 7 M À1 s À1) 6 allows a single ricin molecule to kill a eu-karyotic cell by damaging protein synthesis. 9 The holotoxin is taken up by cells through endocytosis, incorporated into vesicles, and transported …